Preparation of plasmid DNA has become a central protocol in modern molecular biology. Conventional existing techniques for the isolation of plasmid DNA from cells include alkaline lysis, lithium miniprep, boiling miniprep and triton-lysozyme lysis. Such techniques have various limitations such as being time-consuming, complex or simply not suitable for large scale manufacturing processes. Also, these techniques tend to yield an impure DNA product. For example, in the alkaline lysis method, the denatured closed circular form of plasmid DNA formed during the procedure often contaminates the resultant plasmid.
Still other types of methods have also been developed for the isolation of plasmid DNA samples. For example, U.S. Pat. No. 5,561,064 describes a method for producing plasmid DNA which incorporates chromatography for purification. U.S. Pat. No. 5,660,984 describes a method and apparatus for isolating a plasmid DNA sample in which an anion-exchange resin is utilized. U.S. Pat. No. 5,707,812 describes a method for purifying plasmid DNA which also incorporates column chromatography for purification.
Commercial kits have also been specifically designed for the isolation of plasmid DNA and now represent a market worth many millions of dollars per year. For academic laboratories stressed by restricted fiscal resources these kits represent a substantial cost which must be considered against the additional time required for the older, more complex protocols. Most of these kits share a number of steps derived from the alkaline lysis protocol of Bimboim and Doly (1) which has subsequently been improved by the addition of a step in which the DNA is separated from other cellular components while adsorbed to a resin, (usually silica particles). This added step improves the purity of the product by removing some residual protein and most of the residual RNA.
Despite the improved quality of plasmid DNA isolated by existing commercial methods, preparations from many E. coli strains are contaminated by trace amounts of an extremely stable nuclease, namely Endonuclease I (Endo I) and pancreatic ribonuclease introduced in these methods for the purpose of degrading cellular RNA. Endo I, like many other nucleases, is inactive until magnesium is added to the DNA as required by most DNA metabolizing enzymes. The degradation of the plasmid that this enzymatic contamination causes a serious problem if the DNA is to be used in most molecular biology protocols. When recognized the problem may be overcome through the use of an Endo I negative E coli host for the plasmid or through the inclusion of additional steps such as phenol extraction step in the procedure. Such steps such are required whenever it is necessary to transcribe the plasmid since pancreatic ribonuclease, used to reduce contamination of the plasmid by cellular RNA, is sufficiently stable that it can survive the chaotropic solvents used to bind the DNA to the silica matrix.
These modifications to existing conventional protocols are inconvenient. There is no simple selection by which an Endo I deficient mutant of E. coli can be derived from a strain with otherwise desired genotypic characteristics. Phenol extraction is especially time consuming since it requires careful separation of the phases and subsequent complete removal of the phenol. Phenol is also hazardous.
There was therefore a need to develop a reliable, fast, efficient and economical method for the preparation of plasmid nucleic acid that obviated at least one of the limitations of the methods of the prior art.